The development of drug resistance is one of the most perplexing challenges in the field of medicine. One of the most common causes of drug failure in the treatment of diseases involving replicating biological entities, for example, cancer and infectious diseases, is the emergence of drug resistance. One of the most dramatic and tragic examples of drug resistance can be found in connection with the antiviral therapy of acquired immune deficiency syndrome (AIDS).
AIDS is a fatal disease, reported cases of which have increased dramatically within the past several years. Estimates of reported cases in the very near future also continue to rise dramatically.
The AIDS virus was first identified in 1983. It has been known by several names and acronyms. It is the third known T-lymphocyte virus (HTLV-III), and it has the capacity to replicate within cells of the immune system, causing profound cell destruction. The AIDS virus is a retrovirus, a virus that uses reverse transcriptase during replication. This particular retrovirus is also known as lymphadenopathy-associated virus (LAV), AIDS-related virus (ARV) and, most recently, as human immunodeficiency virus (HIV). Two distinct families of HIV have been described to date, namely HIV-1 and HIV-2. The acronym HIV will be used herein to refer to HIV viruses generically.
Specifically, HIV is known to exert a profound cytopathic effect on the CD4+ helper/inducer T-cells, thereby severely compromising the immune system. HIV infection also results in neurological deterioration and, ultimately, in the death of the infected individual.
The field of viral chemotherapeutics has developed in response to the need for agents effective against retroviruses, in particular HIV. For example anti-retroviral agents, such as 3′-azido-2′,3′-dideoxythymidine (AZT), 2′3′-dideoxycytidine (ddC), and 2′3′-dideoxyinosine (ddI) are known to inhibit reverse transcriptase. There also exist antiviral agents that inhibit transactivator protein. Nucleoside analogs, such as AZT, are currently available for antiviral therapy. Although very useful, the utility of AZT and related compounds is limited by toxicity and insufficient therapeutic indices for fully adequate therapy.
Retroviral protease inhibitors also have been identified as a class of anti-retroviral agents. Retroviral protease processes polyprotein precursors into viral structural proteins and replicative enzymes. This processing is essential for the assembly and maturation of fully infectious virions. Accordingly, the design of protease inhibitors remains an important therapeutic goal in the treatment of AIDS.
The use of HIV protease inhibitors, in combination with agents that have different antiretroviral mechanisms (e.g., AZT, ddI and ddT), also has been described. For example, synergism against HIV-1 has been observed between certain C2 symmetric HIV inhibitors and AZT (Kageyama et al., Antimicrob. Agents Chemother., 36, 926-933 (1992)).
Numerous classes of potent peptidic inhibitors of protease have been designed using the natural cleavage site of the precursor polyproteins as a starting point. These inhibitors typically are peptide substrate analogs in which the scissile P1-P1′ amide bond has been replaced by a non-hydrolyzable isostere with tetrahedral geometry (Moore et al, Perspect. Drug Dis. Design, 1, 85 (1993); Tomasselli et al., Int. J. Chem. Biotechnology, 6 (1991); Huff, J. Med. Chem., 34, 2305 (1991); Norbeck et al., Ann. Reports Med. Chem., 26, 141 (1991); and Meek, J. Enzyme Inhibition, 6, 65 (1992)). Although these inhibitors are effective in preventing the retroviral protease from functioning, the inhibitors suffer from some distinct disadvantages. Generally, peptidomimetics often make poor drugs, due to their potential adverse pharmacological properties, i.e., poor oral absorption, poor stability and rapid metabolism (Plattner et al, Drug Discovery Technologies, Clark et al., eds., Ellish Horwood, Chichester, England (1990)).
The design of the HIV-1 protease inhibitors based on the transition state mimetic concept has led to the generation of a variety of peptide analogs highly active against viral replication in vitro (Erickson et al, Science, 249, 527-533 (1990); Kramer et al., Science, 231, 1580-1584 (1986); McQuade et al., Science, 247, 454-456 (1990); Meek et al., Nature (London), 343, 90-92 (1990); and Roberts et al., Science, 248, 358-361 (1990)). These active agents contain a non-hydrolyzable, dipeptidic isostere, such as hydroxyethylene (McQuade et al., supra; Meek et al., Nature (London), 343, 90-92 (1990); and Vacca et al., J. Med. Chem., 34, 1225-1228 (1991)) or hydroxyethylamine (Ghosh et al., Bioorg. Med. Chem. Lett., 8, 687-690 (1998); Ghosh et al., J. Med. Chem., 36, 292-295 (1993)); Rich et al., J. Med. Chem., 33, 1285-1288 (1990); and Roberts et al., Science, 248, 358-361 (1990)) as an active moiety that mimics the putative transition state of the aspartic protease-catalyzed reaction.
Two-fold (C2) symmetric inhibitors of HIV protease represent another class of potent HIV protease inhibitors, which were created by Erickson et al., on the basis of the three-dimensional symmetry of the enzyme active site (Erickson et al. (1990), supra). Typically, however, the usefulness of currently available HIV protease inhibitors in the treatment of AIDS has been limited by relatively short plasma half-life, poor oral bioavailability, and the technical difficulty of scale-up synthesis (Meek et al. (1992), supra).
In a continuing effort to address the problem of short plasma half-life and poor bioavailability, new HIV protease inhibitors have been identified. For example, HIV protease inhibitors incorporating the 2,5-diamino-3,4-disubstituted-1,6-diphenylhexane isostere are described in Ghosh et al., Bioorg. Med. Chem. Lett., 8, 687-690 (1998) and U.S. Pat. No. 5,728,718 (Randad et al.). HIV protease inhibitors, which incorporate the hydroxyethylamine isostere, are described in U.S. Pat. No. 5,502,060 (Thompson et al.), U.S. Pat. No. 5,703,076 (Talley et al.), and U.S. Pat. No. 5,475,027 (Talley et al.).
Recent studies, however, have revealed the emergence of mutant strains of HIV, in which the protease is resistant to the C2 symmetric inhibitors (Otto et al., PNAS USA, 90, 7543 (1993); Ho et al., J. Virology, 68, 2016-2020 (1994); and Kaplan et al., PNAS USA, 91, 5597-5601 (1994)). In one study, the most abundant mutation found in response to a C2 symmetry based inhibitor was Arg to Gln at position 8 (R8Q), which strongly affects the S3/S3′, subsite of the protease binding domain. In this study, the shortening of the P3/P3, residues resulted in inhibitors that were equipotent towards both wild-type and R8Q mutant proteases (Majer et al., 13th American Peptide Symposium, Edmonton, Canada (1993)). Inhibitors have been truncated to P2/P2, without significant loss of activity (Lyle et al., J. Med. Chem., 34, 1230 (1991); and Bone et al., J. Am. Chem. Soc., 113, 9382 (1991)). These results suggest that inhibitors can be truncated and yet maintain the crucial interactions necessary for strong binding. The benefits of such an approach include the elimination of two or more peptide bonds, the reduction of molecular weight, and the diminishment of the potential for recognition by degradative enzymes.
More recently, new mutant strains of HIV have emerged that are resistant to multiple, structurally diverse, experimental and chemotherapeutic retroviral protease inhibitors. Such multidrug-resistant HIV strains are typically found in infected patients, who had undergone treatment with a combination of HIV protease inhibitors or a series of different HIV protease inhibitors. The number of reported cases of patients infected with multidrug-resistant HIV is rising dramatically. Tragically for these patients, the available options for AIDS chemotherapy and/or HIV management is severely limited or is, otherwise, completely nonexistent.
Drug resistance is unfortunately the most common reason for drug failures generally. One of the most dramatic examples of drug failure due to resistance is in HIV therapy. Once HIV resistance is obtained to first-line therapy, the chances of future success are greatly diminished because of the development of multidrug cross resistance. Other diseases involving infectious agents (e.g., viruses, bacteria, protozoa, and prions) or other disease-causing cells (e.g., tumor cells) present similar challenges in that drug resistance is a primary cause of drug failure.
In view of the foregoing problems, there exists a need to determine whether a mutant will be capable of replicating in the presence of a drug. There also exists a need for a method of predicting whether drug resistance is likely to emerge in a disease involving a replicating biological entity. There is also a need for a method of devising a long-term therapeutic regimen that minimizes the likelihood that resistance will occur in a disease involving a replicating biological entity. Moreover, there is a need for a method of preventing or inhibiting the development of drug resistance in such diseases.
The present invention provides such methods. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.